Project Summary The 'immunosandwich' assay is a robust, flexible and sensitive test that is widely employed in clinical diagnostics for monitoring human health and disease. It is based on using pairs of antibodies that recognize different epitopes of the target analyte, thereby providing both sensitivity and specificity. Unfortunately, identifying pairs of antibodies that work in sandwich assays is problematic (i.e., takes time and luck), particularly when they are obtained from animal sources, where there is no specific selection for complementarity. The development cost of a sandwich assay approximates $1M. This technology gap can be filled with recombinant affinity reagents, which are sequenced, renewable, and amenable to protein engineering. Tango Biosciences, Inc. has licensed intellectual property for isolating complementary pairs of affinity reagents, as part of the phage-display affinity selection process, rather than iterative testing post-production. This recently published technology, named MegaSTAR, yields 5 to 10 different pairs of affinity reagents for targets in one month's time. The STTR award will assist in transitioning the technology from an academic center to a commercial entity that can develop a standard operating protocol. Actualization of the company will result from the successful completion of three specific aims. In Specific Aim 1, we will prepare two sets of targets for affinity selection. One set - carcinoembryonic antigen (CEA; biomarker of colorectal cancer) and cardiac troponin T (cTnT; biomarker of myocardial infarction) - will serve to benchmark our reagents against commercial kits that utilize polyclonal and monoclonal antibodies. The second set - leukocyte immunoglobulin-like receptors (LILR) B type (potential immune checkpoint proteins) - will serve as challenging new targets for which monospecific antibodies are not currently available to their ectodomains. In Specific Aim 2, we will use MegaSTAR to generate and optimize pairs of affinity reagents for each target. In Specific Aim 3, we will compare our reagents and commercially available kits for detecting CEA and cTnT in spiked human plasma. The performance metrics (i.e., intra-assay and inter-assay precision, linearity, sensitivity) of reagents will be evaluated through ELISA. Separately, we will evaluate our affinity reagents to the five LILRB ectodomains for their relative specificity through ELISA. Successful completion of the proposed experiments in the 9-month period will enable Tango Biosciences to demonstrate the commercial utility of MegaSTAR and solicit research service contracts from reagent companies and applied markets. The realization of Tango Biosciences? technology platform will positively impact research and clinical testing in ways that potentially improve health treatment and outcomes in the US.